Underlying synaptic transmission is the synaptic vesicle cycle, which involves fusion of synaptic vesicles with the plasma membrane to release neurotransmitter and subsequent retrieval of vesicular proteins via clathrin mediated endocytosis. Clathrin mediated endocytosis is also involved in other processes important for synaptic transmission and the modulation of synaptic strength, including neurotransmitter receptor and transporter trafficking. Clathrin coated vesicles also play major role in trafficking in all compartmentalized cells from yeast to humans. Understanding the mechanisms of clathrin mediated vesicular transport is therefore of broad interest to neuroscientists as well as cell biologists. The clathrin coated vesicle cycle involves recruitment of clathrin triskelia from the cytosol, their polymerization on a membrane to form clathrin coated pits that, following a scission reaction, are internalized as clathrin coated vesicles, and subsequently depolymerized by an Hsp70 chaperone to release triskelia, so that the cycle may continue. Our goals for the next project period will bring a new level of both depth and breadth to our work on this project. As we delve deeper into the synaptic vesicle cycle, we will also broaden the impact of the work, since what we learn about the clathrin coated vesicle and chaperone cycles will impact a wider range of research areas than our initial inquiry into synaptic mechanisms. Indeed, Hsp70 family members play important roles in dissociating the protein aggregates that are associated with many neurodegenerative diseases, so this work will also impact our understanding of protein aggregation disease. Our aims during this next project period are to (1) Characterize the interactions between clathrin and its intrinsically unstructured binding partners. In this aim, we will use NMR spectroscopy to characterize the interaction between the 40 kD N-terminal domain of the clathrin heavy chain and three key endocytic proteins, AP180, AP-2 and amphiphysin. (2) Define the physical mechanism of clathrin coated vesicle uncoating by Hsc70/auxilin. Our work is at the point where we are in a strong position to understand how Hsc70 promotes uncoating at a fundamental level. (3) Define the role of nucleotide exchange factors in regulating Hsc70:clathrin interactions and synaptic vesicle trafficking. We recently discovered that nucleotide exchange factors promote the dissociation of a long-lived Hsc70-clathrin complex. Therefore, in this aim we will determine which nucleotide exchange factor is involved in synaptic vesicle trafficking in nerve terminals, as well as determine how cycles of nucleotide exchange factor:Hsc70 binding and release are controlled. This work is expected to advance our fundamental understanding of the mechanisms that underlie synaptic transmission, and as such will be a critical part of our efforts to fight neurological disorders.